The goal of this project is to determine the molecular mechanisms involved in the assembly of the triad junction between T-tubules and sarcoplasmic reticulum (SR) during the development of excitation- contraction (E-C) coupling in skeletal muscle. Immunofluorescence studies of the distribution of the skeletal muscle dihydropyridine receptor (DHPR) (the putative voltage sensor in E~C coupling), the ryanodine receptor (RyR) (the calcium release channel of the sarcoplasmic reticulum) and triadin in developing normal muscle and dysgenic (mdg) myotubes in culture showed that a protein~protein interaction mediated by the DHPR plays a role in the normal organization of the triad proteins. The alpha1 subunit of the DHPR is essential for the normal targeting of the alpha2 subunit; it also facilitates the normal organization of the RyR and triadin although it is not absolutely required. De novo expression of the DHPR alpha1 subunit from normal nonmuscle nuclei fused with dysgenic myotubes restored normal functions and normal molecular organization of the E~C coupling membranes. Recordings of cytoplasmic free calcium with fluorescent indicators revealed that only action potential~induced calcium transients are eliminated in the dysgenic mutant. Calcium~induced calcium release events were essentially unaltered in the DHPR null mutant, suggesting that these events represent properties of the RyR independent from interactions with the DHPR and from its normal distribution in the junctional face of the terminal SR cisternae. A study of the role of calcium in transcriptional regulation in skeletal myotubes showed that depolarization-induced down- regulation of the acetylcholine receptor alpha subunit is mediated by calcium influx through the DHPR but not by calcium release from the SR. Thus, L~type calcium currents, depolarization~ and calcium~induced calcium all release play different roles in the development and function of skeletal muscle.